WO2017047986A1 - Cultivateur de sphéroïdes cellulaires, et système de culture de sphéroïdes cellulaires le contenant - Google Patents

Cultivateur de sphéroïdes cellulaires, et système de culture de sphéroïdes cellulaires le contenant Download PDF

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Publication number
WO2017047986A1
WO2017047986A1 PCT/KR2016/010128 KR2016010128W WO2017047986A1 WO 2017047986 A1 WO2017047986 A1 WO 2017047986A1 KR 2016010128 W KR2016010128 W KR 2016010128W WO 2017047986 A1 WO2017047986 A1 WO 2017047986A1
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WO
WIPO (PCT)
Prior art keywords
cell spheroid
cell
culture
incubator
spheroid
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Application number
PCT/KR2016/010128
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English (en)
Korean (ko)
Inventor
정초록
정홍렬
김장환
김태돈
손명진
손미영
오수진
임정화
정경숙
조현수
Original Assignee
한국생명공학연구원
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Publication of WO2017047986A1 publication Critical patent/WO2017047986A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/08—Flask, bottle or test tube
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/38—Caps; Covers; Plugs; Pouring means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis

Definitions

  • the present invention relates to a cell spheroid incubator and a cell spheroid culture system including the same, and more particularly, to perform cell spheroid culture more easily, to provide a cell spheroid with high yield, and to produce cell spheroid culture.
  • the present invention relates to a cell spheroid incubator and a cell spheroid culture system including the same, which can further lower the unit cost.
  • Cell culture refers to a process of culturing cells isolated from an organism. Aseptically select biological tissues, treat them with digestive enzymes such as trypsin or pronase, separate them into single cells, perform primary cultures, and transplant single cells obtained by dispersing passaged cell lines or cell lines with the same enzyme treatment to the growth medium. There is a method of dispersing and culturing single cells by treatment with proteolytic enzymes such as inoculation and subsequent passage.
  • the cell culture method is to culture monolayer cells in a two-dimensional flat container. This two-dimensional cell culture and modeling provides key insights into the mechanisms of cell motility and transmission and disease.
  • Three-dimensional cell culture methods in this regard include a hanging-drop method, a rotary culture method, a centrifugal separation method, a micro molding method, and related patents, such as Korean Patent Registration No. 10-1341572 (Modern Culture Method), Korea Patent No. 10-1412155 (rotary culture method), Korea Patent No. 10-1362293 (micro molding method) and the like.
  • the present invention has been made to solve the above problems, the problem of the present invention is to perform the cell spheroid culture more easily, to provide a cell spheroid with a high yield, the cell spheroid culture production cost more It is to provide a cell spheroid incubator and a cell spheroid culture system comprising the same.
  • Cell spheroid incubator has an opening, the body portion containing the cell culture solution, a mesh-shaped net is installed along the inner wall of the body portion, the filter portion is installed inside the body portion detachably from the body portion And is coupled to the opening of the body portion, and includes a cap portion formed with a through hole capable of supplying gas.
  • Cell spheroid culture system has an opening and the body portion containing the cell culture solution, including a mesh-shaped mesh is installed along the inner wall of the body portion and the filter portion is installed inside the body portion to be separated from the body portion,
  • a cell spheroid incubator coupled to an opening of the body part and including a cap part formed with a through hole capable of supplying gas;
  • a rotator having a mounting portion on which the cell spheroid incubator is mounted and rotating the cell spheroid incubator; And a chamber containing a rotator equipped with a cell spheroid incubator.
  • the cell spheroid incubator according to the present invention includes a body portion containing the cell culture solution, a mesh in the form of a mesh, a filter portion installed inside the body portion, and a cap portion coupled to the opening portion and having a through hole capable of supplying gas.
  • the cell spheroid culture system includes a chamber containing a cell spheroid incubator, a rotator for rotating the cell spheroid incubator, and a rotator equipped with the cell spheroid incubator, thereby making cell spheroid culture more convenient.
  • a chamber containing a cell spheroid incubator a rotator for rotating the cell spheroid incubator, and a rotator equipped with the cell spheroid incubator, thereby making cell spheroid culture more convenient.
  • Example 1 is a perspective view showing a cell spheroid incubator according to Example 1 of the present invention.
  • Figure 2 is a perspective view and a partial cross-sectional view for showing the internal structure of the cell spheroid incubator according to Example 1 of the present invention.
  • Figure 3 is a plan view showing a cell spheroid incubator according to Example 1 of the present invention.
  • Figure 4 is a partial cross-sectional view for showing the cap portion of the cell spheroid incubator according to Example 1 of the present invention.
  • Example 5 is a perspective view showing a rotator of a cell spheroid culture system according to Example 2 of the present invention.
  • FIG. 6 is a perspective view showing a chamber of the cell spheroid culture system according to Example 2 of the present invention.
  • Example 7 is a perspective view showing a cell spheroid culture system according to Example 2 of the present invention.
  • Example 8 is a view showing the appearance of the 293 cell spheroid produced through the cell spheroid culture system according to Example 2 of the present invention.
  • FIG. 9 is a view showing the appearance of various other cell spheroids produced through the cell spheroid culture system according to Example 2 of the present invention.
  • FIG 10 is a front view showing the cell spheroid culture system according to Example 3 of the present invention.
  • Example 12 is a right side view showing the cell spheroid culture system according to Example 3 of the present invention.
  • Figure 13 is a conceptual diagram showing the operating mechanism of the cell spheroid culture system according to Example 3 of the present invention.
  • FIG. 1 is a perspective view showing a cell spheroid incubator according to Example 1 of the present invention.
  • Figure 2 is a perspective view and a partial cross-sectional view for showing the internal structure of the cell spheroid incubator according to Example 1 of the present invention.
  • Figure 3 is a plan view showing a cell spheroid incubator according to Example 1 of the present invention.
  • Figure 4 is a partial cross-sectional view for showing the cap portion of the cell spheroid incubator according to Example 1 of the present invention.
  • Example 1 of the present invention a cell spheroid incubator according to Example 1 of the present invention will be described with reference to FIGS. 1 to 4.
  • the cell spheroid incubator 100 includes a body part 110, a filter part 130, and a cap part 150.
  • Body portion 110 has an opening in the upper side, it may be in the form of a tube containing the cell culture.
  • the filter unit 130 may be installed inside the body 110 to be detachably attached to the body 110.
  • the filter unit 130 may include a skeletal member 133 and a mesh 131 having a mesh shape.
  • the skeletal member 133 may be manufactured in a lattice shape, and the mesh net 131 may be installed in a lattice-shaped empty square portion of the skeletal member 133 and may be installed along an inner wall of the body part 110. (See FIG. 2).
  • Mesh-type mesh 131 may be manufactured to have a predetermined eye size as needed. Accordingly, it is possible to determine whether the cell spheroid passes by the predetermined size.
  • the cap 150 may be coupled to the opening of the body 110 to block the opening of the body 110.
  • Cap portion 150 may be coupled (or screwed) to the body portion 110 in the form of a screw, and thus may be more closely coupled.
  • the cap part 150 may include a protrusion 155 protruding in the direction toward the body part 110 (see FIG. 2).
  • the protrusion 155 may serve to fix the filter unit 130 by fitting the filter unit 130 inward.
  • the cap part 150 when the cap part 150 is coupled to the body part 110, the cap part 150 not only serves to block the opening of the body part 110, but also tightly grips the filter part 130 by the protrusion 155. Accordingly, shaking of the filter unit 130 may be prevented and the filter unit 130 may be firmly fixed.
  • At least one through hole 160 may be formed in the cap part 150 to enable gas supply to the inside of the body part 110.
  • the inflow of external gas can be controlled through the through hole 160.
  • the through hole 160 may serve as a passage for allowing air to enter and exit the body 110 when necessary in the culture process.
  • the cap part 150 includes a second cap part 153 rotatably coupled to the first cap part 151 and the first cap part 151 about the central axis O. It may include.
  • the first cap part 151 may have a plurality of through holes of different sizes.
  • the through holes 161 of the four first cap portions which increase in size as the clockwise goes, are illustrated.
  • the second cap part 153 may have a through hole formed at a position corresponding to the through hole formed in the first cap part 151.
  • the through hole 163 of the second cap portion having a corresponding size is shown at a position corresponding to the four through holes of the first cap portion 151.
  • the number of the through holes opened may be adjusted in such a way that some of the four through-holes 161 of the first cap part are opened and a part thereof is blocked.
  • the amount of gas supplied to the inside can be adjusted. For example, when culturing cells under conditions such as hypoxic conditions, the inflow of oxygen may be lowered by reducing the degree of opening and closing of the through hole 160 provided in the cap 150.
  • the through hole 160 provided in the cap part 150 may be a passage through which air may enter and exit the cell, and at this time, a filtration filter (not shown) may be installed inside the through hole. Filtration filter prevents the microorganisms contained in the air from entering the culture medium through the air passage, thereby allowing only air to enter and exit.
  • the filter unit 130 is wrapped in a mesh net 131 is coupled within the body portion 110 can prevent the cells of different sizes from being mixed between the inside and the outside of the filter unit 130. .
  • the filter unit 130 has a mesh form of a mesh
  • the cell spheroid formed inside the filter unit 130 is located inside the filter unit 130 and cannot go out, but the size is small.
  • the cells may be mixed into and out of the filter unit 130.
  • the filter unit 130 of the cell spheroid incubator 100 may be easily separated from the body unit 110. Therefore, it can be very convenient to separate only the filter unit 130 formed with the three-dimensional cell spheroid to move to other culture conditions. In addition, the separation of the cell spheroids and single cells that do not form a spheroid may be easy.
  • the cell spheroid incubator 100 includes a body part 110 containing a cell culture solution and a filter part 130 to form a three-dimensional cell spheroid inside the body part 110. And, by having a cap 150 to block the opening of the body portion 110 to control the inflow of external gas, it is possible to perform the three-dimensional cell spheroid culture more easily, such as significantly simplifying the mounting and recovery process of the cells Can provide cell spheroids in high yield, and can further lower the cost of producing cell spheroid cultures.
  • 5 is a perspective view showing a rotator of a cell spheroid culture system according to Example 2 of the present invention.
  • 6 is a perspective view showing a chamber of the cell spheroid culture system according to Example 2 of the present invention.
  • 7 is a perspective view showing a cell spheroid culture system according to Example 2 of the present invention.
  • Cell spheroid culture system according to Example 2 of the present invention comprises a cell spheroid incubator according to Example 1 described above.
  • Example 2 is different from Example 1 in that it further includes a rotator and a chamber.
  • Example 2 of the present invention a cell spheroid culture system according to Example 2 of the present invention will be described with reference to FIGS. 5 to 7.
  • the cell spheroid culture system 1000 includes the cell spheroid incubator 100, the rotator 210, and the chamber 230 according to the first embodiment.
  • the rotator 210 may include a mounting portion 211 on which the cell spheroid incubator 100 is mounted, and rotate the cell spheroid incubator 100.
  • the rotator 210 may rotate the cell spheroid incubator 100 in the R direction and adjust the rotation speed thereof.
  • the chamber 230 may accommodate the rotator 210 equipped with the cell spheroid incubator 100 in the inner space 231 of the chamber.
  • the chamber 230 may be made of acrylic or another material, and may withstand a pressure of 1 bar and maintain a predetermined pressure by adjusting to a desired pressure.
  • the chamber 230 may adjust an internal temperature, and may constantly control the cell culture conditions of 37 degrees Celsius.
  • the chamber 230 may also have waterproofing means to prevent unnecessary liquids from penetrating into the chamber 230.
  • the chamber 230 may be installed outside the chamber 230 and may include a controller 233 for adjusting the rotational speed of the rotator 210 accommodated in the chamber 230 (see FIG. 7).
  • the controller 233 is connected to the rotator 210 in a wired or wireless manner so as to transmit and receive an electrical signal with the rotator 210, and thus the chamber 230 outside the chamber 230 without opening the chamber 230. Rotational speed of the cell spheroid incubator 100 inside) can be adjusted as needed.
  • the cumbersomeness can be reduced, and the rotation speed of the cell spheroid incubator 100 is controlled without opening the chamber 230, thereby preventing the environment in which the cells are cultured inside the chamber 230. May not be received and thus the production efficiency and productivity of the cell spheroids may be significantly improved.
  • the cell spheroid culture system 1000 can perform cell spheroid culture more easily, provide a cell spheroid with high yield, and further lower the cell spheroid culture production cost. Can be.
  • FIG 8 is a view showing the appearance of the 293 cell spheroid produced through the cell spheroid culture system 1000 according to Example 2 of the present invention.
  • 9 is a view showing the appearance of various other cell spheroids produced through the cell spheroid culture system 1000 according to the second embodiment of the present invention.
  • a culture solution containing 293 cells was accommodated in the cell spheroid incubator 100, installed in the rotator 210, and rotated at 5 rpm, and placed in a chamber 230 at 37 ° C., 5%, and CO 2 to incubate the cells. .
  • the state of the spheroid produced was observed through an optical microscope.
  • the 293 cell spheroid thus produced is shown in FIG. 8.
  • FIG. 10 is a front view showing the cell spheroid culture system according to Example 3 of the present invention.
  • Fig. 11 is a left side view showing the cell spheroid culture system according to Example 3 of the present invention.
  • 12 is a right side view showing the cell spheroid culture system according to Example 3 of the present invention.
  • FIG. 13 is a conceptual diagram showing an operating mechanism of the cell spheroid culture system 3000 according to Embodiment 3 of the present invention.
  • Example 3 differs from Example 2 in that the chamber comprises a control unit and a culture unit.
  • the same (or substantial) reference numerals will be given to the same (or substantial) parts as those described above, and detailed description thereof will be omitted.
  • Example 3 of the present invention a cell spheroid culture system according to Example 3 of the present invention will be described with reference to FIGS. 10 to 13.
  • a chamber in the cell spheroid culture system 3000 according to Embodiment 3 of the present invention, includes a controller 333 and a culture unit 335.
  • the culture unit 335 is a portion in which the rotator 310 is accommodated, and may mean a portion in which cells are cultured.
  • the control unit 333 may have a feature that is installed in isolation from the culture unit 335. Accordingly, the speed control of the rotator 310 inside the culture unit 335 can be performed outside the culture unit 335. Therefore, the speed of the rotator 310 inside the culture unit 335 can be adjusted without opening and closing the inlet of the culture unit 335 to the outside. As a result, the cell culture environment inside the culture unit 335 may be smoothly performed without being disturbed.
  • the cell culture environment inside the culture unit 335 may be affected by the external environment to provide an appropriate cell culture environment. You will not be able to maintain it. In this case, the efficiency of cell culture may be reduced.
  • the rotator 310 installed in the culture unit 335 may include a motor 313 and a rotation shaft connected to the motor 313 (see FIG. 13).
  • the mounting part 311 may be installed on the rotating shaft (see FIG. 11).
  • the cell spheroid incubator 100 may be installed in the mounting portion 311.
  • the mounting portion 311 rotates, and accordingly, the spheroid incubator 100 also rotates.
  • the controller 333 may include a control connector 343 and a main controller 341.
  • the control connector 343 may be electrically connected to the motor 313 inside the rotator 310 to recognize the rotational speed of the motor 313.
  • the main controller 341 may be connected to the control connector 343 to exchange electrical signals with the control connector 343, and may control the rotational speed of the motor identified from the control connector 343.
  • the control connector 343 may recognize this and transfer this information to the main controller 341.
  • the main controller 341 may transmit a signal to the motor 313 to make the speed of the motor 313 higher, and the motor 313 receiving this signal may speed up the rotation speed of the mounting part 311. can do.
  • the main controller 341 may also be connected to the other temperature sensor 345 and the pressure sensor 347 to control the temperature and pressure inside the culture unit 335. In this case, the main controller 341 may simultaneously control the rotation speed of the rotator 310, the motor 313, the temperature inside the culture unit 335, and the pressure inside the culture unit 335. In this case, the environment inside the culture unit 335 may be a more suitable environment for cell spheroid culture.

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Abstract

La présente invention concerne un cultivateur de sphéroïdes cellulaires, et un système de culture de sphéroïdes cellulaires le contenant et, plus spécifiquement, un cultivateur de sphéroïdes cellulaires qui peut conduire, de manière plus pratique, la culture de sphéroïdes cellulaires, fournir un sphéroïde cellulaire sous haut rendement, et en outre réduire les coûts de production de la culture de sphéroïdes cellulaires, et un système de culture de sphéroïdes cellulaires le contenant. Selon la présente invention, le cultivateur de sphéroïdes cellulaires comprend : une partie formant corps ayant une ouverture et contenant une solution de culture de cellules ; une partie formant filtre comprenant un filet à mailles disposé le long de la paroi interne de la partie formant corps, et prévue à l'intérieur de la partie formant corps afin d'être capable d'être séparée de la partie formant corps ; et une partie formant bouchon accouplée à l'ouverture de la partie formant corps, et présentant un trou traversant pour l'alimentation en gaz.
PCT/KR2016/010128 2015-09-18 2016-09-09 Cultivateur de sphéroïdes cellulaires, et système de culture de sphéroïdes cellulaires le contenant WO2017047986A1 (fr)

Applications Claiming Priority (2)

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KR1020150132656A KR101743231B1 (ko) 2015-09-18 2015-09-18 세포 스페로이드 배양기를 포함하는 세포 스페로이드 배양 시스템
KR10-2015-0132656 2015-09-18

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
GB2590434A (en) * 2019-12-17 2021-06-30 Oribiotech Ltd An apparatus
US11732232B2 (en) 2020-08-14 2023-08-22 Korea Research Institute Of Bioscience And Biotechnology Biomimetic cell culture apparatus and cell culture system comprising the same

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KR102156310B1 (ko) 2019-03-06 2020-09-15 울산과학기술원 바이오잉크를 이용한 세포 스페로이드 제조 방법
KR102303845B1 (ko) 2019-10-14 2021-09-17 연세대학교 산학협력단 멀티 레이어 스페로이드 배양장치
KR102391692B1 (ko) * 2020-08-04 2022-04-28 성균관대학교산학협력단 스페로이드 제너레이터, 스페로이드 배양키트 및 스페로이드 배양방법

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JP2010168122A (ja) * 1999-10-20 2010-08-05 Qiagen North American Holdings Inc 混合・注入装置及びその容器
JP2007312730A (ja) * 2006-05-29 2007-12-06 Stanley Electric Co Ltd 組織培養播種装置。
JP2009118820A (ja) * 2007-11-19 2009-06-04 Niigata Univ コンパクト回転培養システム及び培養容器
KR20140091270A (ko) * 2013-01-11 2014-07-21 주식회사 에스피엘 세포배양용 캡 조립체 및 이를 포함하는 세포배양용 용기
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2590434A (en) * 2019-12-17 2021-06-30 Oribiotech Ltd An apparatus
GB2590434B (en) * 2019-12-17 2022-02-16 Oribiotech Ltd An Apparatus
US11732232B2 (en) 2020-08-14 2023-08-22 Korea Research Institute Of Bioscience And Biotechnology Biomimetic cell culture apparatus and cell culture system comprising the same

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KR20170034241A (ko) 2017-03-28

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